Methods and compositions for detection of microbial contaminants in peritoneal dialysis solutions

ABSTRACT

Methods and compositions for detection of microbial contaminants in peritoneal dialysis solutions are provided. A novel cause of aseptic peritonitis is provided—aseptic peritonitis associated with gram positive microbial contamination of a dialysis solution. Peptidoglycan is a major component of a gram positive bacterial cell wall and thus can serve as a marker for gram positive bacteria. In this regard, testing for peptidoglycans can be utilized to effectively prevent peritonitis in patients that use the peritoneal dialysis solutions, such as peritoneal dialysis solutions that contain a glucose polymer including an icodextrin and the like.

BACKGROUND OF THE INVENTION

The present invention generally relates to the detection of grampositive microbial contaminants. More specifically, the presentinvention relates to methods and compositions that employ the detectionof peptidoglycan in peritoneal dialysis solutions. Peptidoglycans aremajor cell wall components of gram positive organisms and thus serve asa good marker of these microbes.

Due to disease or insult or other causes, the renal system can fail. Inrenal failure of any cause, there are several physiologicalderangements. The balance of water, minerals (e.g., Na, K, Cl, Ca, P,Mg, SO₄) and the excretion of a daily metabolic load of fixed ions is nolonger possible in renal failure. During renal failure, toxic endproducts of nitrogen metabolism (e.g., urea, creatinine, uric acid, andthe like) can accumulate in blood and tissues.

Dialysis processes have been devised for the separation of elements in asolution by diffusion across a semi-permeable membrane (diffusive solutetransport) across a concentration gradient. Examples of dialysisprocesses include hemodialysis, peritoneal dialysis and hemofiltration.

Hemodialysis treatment utilizes the patient's blood to remove waste,toxins, and excess water from the patient. The patient is connected to ahemodialysis machine and the patient's blood is pumped through themachine. Catheters or the like are inserted into the patient's veins andarteries to connect the blood flow to and from the hemodialysis machine.Waste, toxins, and excess water are removed from the patient's blood andthe blood is infused back into the patient. Hemodialysis treatments canlast several hours and are generally performed in a treatment centerabout three or four times per week.

To overcome the disadvantages often associated with classicalhemodialysis, other techniques were developed, such as peritonealdialysis.

Peritoneal dialysis utilizes the patient's own peritoneum as asemipermeable membrane. The peritoneum is the membranous lining of thebody cavity that, due to the large number of blood vessels andcapillaries, is capable of acting as a natural semipermeable membrane.

In peritoneal dialysis, a sterile dialysis solution is introduced intothe peritoneal cavity utilizing a catheter or the like. After asufficient period of time, an exchange of solutes between the dialysateand the blood is achieved. Fluid removal is achieved by providing asuitable osmotic gradient from the blood to the dialysate to permitwater outflow from the blood. This allows a proper acid-base,electrolyte and fluid balance to be returned to the blood. The dialysissolution is simply drained from the body cavity through the catheter.Examples of different types of peritoneal dialysis include continuousambulatory peritoneal dialysis, automated peritoneal dialysis andcontinuous flow peritoneal dialysis.

Standard peritoneal dialysis solutions contain dextrose to effecttransport of water and metabolic waste products across the peritoneum.Although dextrose has the advantage of being relatively safe andinexpensive, it has a number of disadvantages. Because of the smallsize, dextrose is rapidly transported through the peritoneum, thusleading to the loss of osmotic gradient and loss of ultrafiltrationwithin about 2 to 4 hours of infusion. It has been suggested that theultrafiltration characteristics of peritoneal dialysis solutions couldbe improved by replacing dextrose with large molecular weightsubstances, such as glucose polymers. An example of a novel highmolecular weight agent is icodextrin. Dialysis solutions containingicodextrin are commercially available and have been found to be usefulin treating patients with end stage renal disease.

Peritonitis is a major complication of peritoneal dialysis. Clinicalsuspicion of peritonitis is prompted by the development of acloudy-appearing dialysate in combination with variable clinicalmanifestations that may include abdominal pain, nausea, vomiting,diarrhea and fever. See, for example, Vas S I: Peritonitis. In: Nolph KD, ed. Peritoneal Dialysis. 3^(rd) ed. Dordrecht, The Netherlands:Kluwer Academic Publishers, 1989:261-84. Most episodes of peritonitisare caused by intraperitoneal bacterial infections, and the diagnosis isusually readily established by positive dialysate cultures. However,there are several well documented causes of non-infectious or sterileperitonitis. Aseptic or sterile peritonitis, which also is described asaseptic, chemical, or culture-negative peritonitis, is typically causedby a chemical or a foreign body irritant.

One of the major outbreaks of sterile peritonitis among patients onperitoneal dialysis occurred in 1977. This was attributed to intrinsicand occult endotoxin contamination of dialysis solution. Suspectedprovocative batches of peritoneal dialysate had endotoxin levels in therange of 2 to 2.5 endotoxin units (EU)/mL. See, for example, KaranicolasS, Oreopoulos D G, Izatt S H, et al: Epidemic of aseptic peritonitiscaused by endotoxin during chronic peritoneal dialysis, N Engl J Med1977; 296:1336-7. A similar epidemic of aseptic peritonitis caused byendotoxin contamination in continuous cycling peritoneal dialysispatients was reported in 1998. See, for example, Mangram A J, Archbald LK, Hupert M, et al: Outbreak of sterile peritonitis among continuouscycling peritoneal dialysis patients, Kidney Int 1988; 54:1367-71. Otherreported causes of aseptic peritonitis include intraperitonealadministered vancomycin (See, for example, Smith T, Baile G, Eisele G:Chemical peritonitis associated with intraperitoneal vancomycin, AnnPharm 1991; 25:602-3, and Chancy D I, Gouse S F: Chemical peritonitissecondary to intraperitoneal vancomycin, Am J Kidney Dis 1991; 17:76-9),amphotericin B (See, for example, Benevent D, El Akoun N, Lagarde C:Dangers of administration of intraperitoneal amphotericin B incontinuous ambulatory peritoneal dialysis, Press Med 1984; 13:1844), andacetaldehyde (See, for example, Tuncer M, Sarikaya M, Sezer T, et al:Chemical peritonitis associated with high dialysate acetaldehydeconcentrations, Nephrol Dial Transplant 2000; 15:2037-40). A unique formof aseptic peritonitis, eosinophilic peritonitis, is a much more commonentity that can occur shortly after the start of peritoneal dialysis.See, for example, Gokal R, Ramos J M, Ward M K, et al: Tosinophilicperitonitis' in CAPD, Clin Nephrol 1981; 15:328-330.

As previously discussed, glucose polymers, such as icodextrin, can beused in place of dextrose in peritoneal dialysis solutions. Icodextrinis a polymer of glucose derived from the hydrolysis of corn starch. Ithas a molecular weight of 12-20,000 Daltons. Peritoneal dialysissolutions containing icodextrin as the osmotic agent are, in general,used for long dwell (>4 hour) exchanges. The majority of glucosemolecules in icodextrin are linearly linked with α(1-4) glucosidic bonds(>90%) while a small fraction (<10%) is linked by α(1-6) bonds.

Icodextrin was introduced into clinical practice in the United Kingdomin 1994 and in other European countries beginning in 1996. The clinicaladvantages of icodextrin for long dwells, especially in patients withhigh and high average transport status and loss of ultrafiltration, iswell-accepted and contributed to its global popularity. See, forexample, Wilkie M E, Plant M J, Edwards L, et al: Icodextrin 7.5%dialysate solution (glucose polymer) in patients with ultrafiltrationfailure: extension of technique survival, Perit Dial Int 1997; 17:84-7;Wolfson M, Piraino B, Hamburger R J, Morton A R, for the IcodextrinStudy Group: A randomized controlled trial to evaluate the efficacy andsafety of icodextrin in peritoneal dialysis, Am J Kidney Dis 2002;40:1055-65; and Mujais S, Nolph K, Gokal R, et al: Evaluation andmanagement of ultrafiltration problems in peritoneal dialysis, PeritDial Int 2000; 20(Suppl 4):S5-S21.

Since the introduction of icodextrin for use in peritoneal dialysissolutions, sporadic cases of aseptic peritonitis have been reported.See, for example, Pinerolo M C, Porri M T, D'Amico G: Recurrent sterileperitonitis at onset of treatment with icodextrin, Perit Dial Int 1999;19:491-2; Williams P F: Timely initiation of dialysis. Am J Kidney Dis34:594-595, 1999; Williams P F, Foggensteiner L: Sterile/allergicperitonitis with icodextrin in CAPD patients, Perit Dial Int 2002;22:89-90; Foggensteiner L, Bayliss J, Moss H, et al: Timely initiationof dialysis—single-exchange experiences in 39 patients startingperitoneal dialysis, Perit Dial Int 2002; 22:471-6; Heering P, Brause M,Plum J, et al: Peritoneal reaction to icodextrin in a female patient onCAPD. Perit Dial Int 2001; 21:321-2; Del Rosso G, Di Liberato L, PirilliA, et al: A new form of acute adverse reaction to icodextrin inperitoneal dialysis patient, Nephrol Dial Transplant 2000; 15:927-8;Goffin E, Scheiff J M: Transient sterile chemical peritonitis in a CAPDpatient using icodextrin, Perit Dial Int 2002; 22:90-1; Tintillier M,Pochet J M, Christophe J L, Scheiff J M, et al: Transient sterilechemical peritonitis with icodextrin: clinical presentation, prevalence,and literature review, Perit Dial Int 2002; 22:534-7; and Gokal R:Icodextrin-associated sterile peritonitis, Perit Dial Int 2002;22:445-8. These patients typically presented with cloudy dialysate, noabdominal pain, and dialysate cell counts varying from 300 to 3500/mm³,with variable percentages of neutrophils, lymphocytes, and macrophages.In general, there is no change in ultrafiltration profile or peritonealpermeability for solutes. Cultures were invariably negative with noevidence of peritoneal or peripheral blood eosinophilia. Moreover, allsolution components and endotoxin levels fell within the productspecifications, and the icodextrin-based peritoneal dialysis solutionsmet all current Pharmacopoeia standards. Prompted by these reports, in2001, the manufacturer of the icodextrin-containing solution (BAXTERHEALTHCARE CORPORATION) modified the Summary of Product Characteristics(SPC) to include cloudy effluent as an “undesirable side effect” oficodextrin.

Parenteral pharmaceutical products are required to be free ofcontaminating substances, such as substances that can cause fever.Because endotoxins derived from gram-negative bacteria are the mostcommon contaminant in parenteral products, the historic pyrogens ofconcern are LPS. Current Pharmacopeia standards are that one of twotests for pyrogenic contamination is applied to parenteral products.These tests are the rabbit pyrogen test and the LAL assay. Althoughgenerally reliable, both tests have shortcomings. The rabbit test relieson a febrile response that in turn depends upon the elaboration ofpyrogenic cytokines. The rabbit pyrogen testing may be falsely negative,if the pyrogen is at a concentration too low to induce a systemicresponse, but of sufficient magnitude to produce a local inflammatoryreaction. In turn, the more sensitive LAL test does not detect pyrogensother than LPS. Pyrogens, like viruses, fungi, DNA, gram-positiveexotoxins, or bacterial cell wall components from gram positivebacteria, such as peptidoglycan and the like, will not be detected bythe LAL test. See, for example, Dinarello C A, O'Conner J V, LoPreste G:Human leukocyte pyrogen test for detection of pyrogenic material ingrowth hormone produced by recombinant Escherichia coli, J ClinMicrobiol 1984; 20:323-9; Poole S, Thorpe R, Meager A, et al: Detectionof pyrogen by cytokine release, Lancet 1988; 1(8577):130; Ray A, RedheadK, Selkirk S, et al: Variability in LPS composition, antigenicity andreactogenicity of phase variants of Bordetella pertussis, FEMS MicrobiolLett 1991; 63:211-7; Taktak Y S, Selkirk S, Bristow A F, et al: Assay ofpyrogens by interleukin-6 release from monocytic cell lines, J PharmPharmacol 1991; 43:578-82; and Fennrich S, Fischer M, Hartung T, et al:Detection of endotoxins and other pyrogens using human whole blood, DevBiol Stand 1999; 101:131-9.

The global outbreak of aseptic peritonitis observed withicodextrin-based peritoneal dialysis solutions as discussed above servesas a sentinel example of how contemporary parenteral products withmicrobial, non-endotoxin contaminants may be considered safe underPharmacopoeia standards but provoke adverse clinical effects. Therefore,a need exists to provide improved standards for parenteral products thatemploy detection procedures to better ensure that the parenteralproducts are effectively free of contaminating substances.

SUMMARY OF THE INVENTION

The present invention generally relates to the detection of grampositive microbial contaminants. In particular, the present inventionrelates to methods and compositions that employ the detection ofpeptidoglycan in peritoneal dialysis solutions. The inventors havesurprisingly discovered a novel cause of aseptic peritonitis—asepticperitonitis associated with gram positive microbial contamination of adialysis solution. Peptidoglycan is a major component of a gram positivebacterial cell wall and thus can serve as a marker for gram positivebacteria. Thus, testing for peptidoglycans can be utilized toeffectively prevent peritonitis in patients that use the peritonealdialysis solutions, such as peritoneal dialysis solutions that contain aglucose polymer including an icodextrin and the like.

Icodextrin is derived from corn starch, a natural product. It is wellknown that products of natural origin are contaminated with a widevariety of micro-organisms. The inventors have found that some naturalproducts, such as corn starch, contain an acidophilic thermophilicbacteria, such as Alicyclobacillus acidocaldariuos. The later organismis ubiquitous in the food industry, particularly in acidic beverages. Itis the alicyclobacillus that produces guaiacol, which is a causativesubstance for an ‘off’ flavour orange juice. See, for example, MatsubaraH, Goto K, Matsumara T, et al. Alicyclobacillus acidiphilus sp. Nov., anovel thermo-acidophilic, omega-alicyclic fatty acid-containingbacterium isolated from acidic beverages. Int J Syst Evol Microbiol2002; 52:1681-5.

Peritoneal dialysis solutions and parenteral solutions in general havenot been recognized to have contaminated by this organism or itsdegradation products. This is mainly because the current testingprocedure for microbial contamination of peritoneal dialysis solutionsand parenteral solutions in general are not capable of detecting thisorganism or its degradation products.

To this end, in an embodiment, the present invention provides a methodfor manufacturing a peritoneal dialysis solution. The method includesproviding a glucose polymer-based solution; adding a reagent to theglucose polymer-based solution wherein the reagent is capable ofreacting with a peptidoglycan; determining an amount of thepeptidoglycan using the reagent; and using the glucose polymer-basedsolution to provide the peritoneal dialysis solution if it is determinedthat a sufficiently low level of the peptidoglycan is present.

In an embodiment, the reaction initiates a serine protease cascade.

In an embodiment, the serine protease cascade includes a prophenoloxidase cascade.

In an embodiment, the reagent is derived from a silkworm larvae plasma.

In an embodiment, the amount of peptidoglycan is further determined by acolorimetric measurement in response to the reaction between thepeptidoglycan and the reagent.

In an embodiment, the sufficiently low level of the peptidoglycan isabout 10 ng/mL or less.

In an embodiment, the glucose polymer-based solution includes anicodextrin.

In an embodiment, the reagent is added to the icodextrin in a rawmaterial form that is used to make the glucose polymer-based solution.

In another embodiment, the present invention provides a method ofproviding peritoneal dialysis to a patient. The method includespreparing a peritoneal dialysis solution utilizing a reagent to ensurethat the peritoneal dialysis solution has a sufficiently low level of apeptidoglycan so as to prevent peritonitis in the patient; and providingthe peritoneal dialysis solution to the patient.

In an embodiment, the peritoneal dialysis solution includes anicodextrin-based solution.

In an embodiment, the peritoneal dialysis includes an automatedperitoneal dialysis, a continuous ambulatory peritoneal dialysis and thelike.

In an embodiment, the patient is monitored for peritonitis duringperitoneal dialysis. For example, a dialysis effluent can be collectedfrom the patient to determine an IL-6 response that correlates to anincidence of peritonitis.

In an embodiment, the reagent is used to determine if the amount of thepeptidoglycan exceeds about 10 ng/mL in the peritoneal dialysis solutionprior to use during peritoneal dialysis.

In yet another embodiment, the present invention provides a method oftesting a peritoneal dialysis solution for an amount of a peptidoglycanthat exceeds a level sufficient to cause peritonitis. The methodincludes adding a reagent to the peritoneal dialysis solution whereinthe reagent is capable of reacting with the peptidoglycan to initiate aserine protease cascade; and determining the amount of thepeptidoglycan. For example, the icodextrin-based solution can be testedto determine if the amount of peptidoglycan exceeds about 10 ng/mL.

In still yet another embodiment, the present invention provides anicodextrin composition that includes a reagent capable of reacting witha peptidoglycan.

In an embodiment, a level of peptidoglycan can be established abovewhich the product would produce sterile peritonitis.

In an embodiment, use of affinity columns with resins that bindpeptidoglycan can be used to minimize the contamination of peptidoglycanin glucose polymers.

An advantage of the present invention is to provide improved peritonealdialysis solutions.

Another advantage of the present invention is to provide improvedmethods for manufacturing and using peritoneal dialysis solutions thatemploy a detection protocol to determine the presence of peptidoglycanin the peritoneal dialysis solution.

Yet another advantage of the present invention is to provide improvedtesting procedures that can be employed to prevent peritonitis inpatients that receive peritoneal dialysis therapy.

Yet still another advantage of the present invention is to provide animproved icodextrin composition that utilizes a detection procedure inthe manufacture thereof to determine the presence of peptidoglycan.

Additional features and advantages of the present invention aredescribed in, and will be apparent from, the following DetailedDescription of the Invention and the figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a correlation between IL-6 response in a PBMC assayand peptidoglycan concentration in icodextrin. IL-6 response wasmeasured in freshly isolated monocytes from healthy volunteers. Eachsymbol and line represent data from a single donor.

FIG. 2 illustrates an effect of peptidoglycan on the infiltration ofneutrophils in the peritoneal fluid of rats. Peritoneal fluid wascollected six hours after single injection (35 mL/kg) of icodextrincontaining peptidoglycan.

FIG. 3 illustrates an effect of peptidoglycan on TNF-α in the peritonealfluid of rats.

FIG. 4 illustrates an effect of peptidoglycan on IL-6 in the peritonealfluid of rats.

FIG. 5 illustrates frequency (%) of aseptic peritonitis reported withuse of icodextrin.

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally relates to the detection of grampositive organisms and fragments thereof. In particular, the presentinvention relates to methods and compositions that employ the detectionof peptidoglycan in peritoneal dialysis solutions. The inventors havesurprisingly discovered a novel cause of aseptic peritonitis-asepticperitonitis associated with gram positive microbial contamination of adialysis solution. Peptidoglycan is a major component of a gram positivebacterial cell wall and thus can serve as a marker for gram positivebacteria. In this regard, testing for peptidoglycans can be utilized toeffectively prevent peritonitis in patients that use the peritonealdialysis solutions, such as peritoneal dialysis solutions that contain aglucose polymer including an icodextrin and the like.

Aseptic peritonitis associated with icodextrin-based peritoneal dialysissolutions is believed to be the largest adverse event reported for aperitoneal dialysis solution due to a contaminant of microbial origin.Based on the experimental investigations as described in detail below,this suggests that peptidoglycan in the icodextrin-based peritonealdialysis solution was the causative agent of aseptic peritonitis.Further, pharmacovigilence data as detailed below supports theeffectiveness of the corrective action and manufacturing screeningprocedure to prevent the occurrence of peritonitis. These findingsillustrate that while endotoxin is deservedly one of the more worrisomebacterial product that can cause adverse effects to patients, it is notthe sole one. In this regard, non-endotoxin pyrogens, such aspeptidoglycans, that have not been previously identified, are capable ofproducing clinically significant inflammation. Thus, this demonstratesthat parenteral pharmaceutical products that pass the compendial testsand so meet Pharmacopoeia standards may still require a further level oftesting to effectively determine the efficacy and safe use of suchproducts to better ensure quality of life issues associated with use ofsame.

In the present invention, non-LPS pyrogen contamination was recognizedas a problem because peritoneal dialysis exchanges permitted the directobservation of peptidoglycan-induced inflammation in situ. Peptidoglycanis a heteropolymer formed from β (1-4) linked N-acetylmuramic acid andN-acetyl-D-glucosamine residues crosslinked by peptide bridges. See, forexample, Royce C L, Pardy R L: Endotoxin-like properties of an extractfrom a symbiotic, eukaryotic cholerella-like green algae, J EndotoxinRes 1996; 3:437-44. The glycan backbone is chemically homogenous,whereas the peptides cross-linking the sugars vary. Peptidoglycanoccupies approximately 40% by weight of gram positive cell wall, butabout 1-10% of the total weight of the gram negative cell walls. Royce CL, Pardy R L: Endotoxin-like properties of an extract from a symbiotic,eukaryotic cholerella-like green algae, J Endotoxin Res 1996; 3:437-44.Peptidoglycan and another cell wall constituent, lipoteichoic acid,include virtually all of the major inflammatory inducing components ofgram-positive cellular walls. See, for example, Sriskandan S, Cohen J:Gram-positive sepsis, In: Opal S M, Cross A S, eds. Bacterial Sepsis andSeptic Shock, Philadelphia: W. B. Saunders Company, 1999:397-412.

Like endotoxins, peptidoglycans can induce cytokine production in a widevariety of cells and have long been recognized to have immunomodulatoryactions. See, for example, Garner R E, Hudson J A: Intravenous injectionof candida-derived mannan results in elevated tumor necrosis factoralpha levels in serum, Infect Immun 1996; 64:4561-6; and Schwab J:Phlogistic properties of peptidoglycan-polysaccharide polymers from cellwalls of pathogenic and normal flora bacteria which colonize humans,Infect Immun 1993; 61:4535-9. However, peptidoglycans are several ordersof magnitude less potent than endotoxins as provocateurs of thesebiological effects. See, for example, Henderson B, Poole S, Wilson M:Bacterial modulins: a novel class of virulence factors which cause hosttissue pathology by inducing cytokine synthesis, Microbiol Rev 1996;60:316-41; and Nakagawa Y, Maeda H, Murai T: Evaluation of the in vitropyrogen test system based on proinflammatory cytokine release from humanmonocytes: Comparison with a human whole blood culture test system andwith the rabbit pyrogen test, Clin Diag Lab Immunol 2002; 9:588-97. Forexample, the minimum pyrogenic dose of peptidoglycans in rabbits is 7.3pg/kg, while that of endotoxins is 0.0027 μg/kg. See, for example,Henderson B, Poole S, Wilson M: Bacterial modulins: a novel class ofvirulence factors which cause host tissue pathology by inducing cytokinesynthesis, Microbiol Rev 1996; 60:316-41.

In addition to the absence of pyrogenic containing substances, safety ofparenteral products are generally defined by Pharmacopoeia tests todetermine their sterility. Bacterial cultures are generally performed atneutral pH using an incubation temperature between 20-35° C. These aresuboptimal conditions for the growth of thermophilic, acidophilicmicroorganisms such as Alicyclobacillus acidocaldarius that require anacid medium and elevated temperature for growth. Therefore, routinelyemployed “sterility definitions” and the supporting assays may fail todetect microorganisms that do not grow under conventional conditions. Inan embodiment of the present application, acid hydrolysis at an elevatedtemperature is used for hydrolysis of starch to produce icodextrin.These manufacturing conditions are suitable for the growth ofAlicycclobacillus acidocadarius, but discordant with those used todetermine sterility based on bioburden.

As referenced above, a detailed description of the investigativefindings are provided below in accordance with an embodiment of thepresent invention by way of example and without limitation:

Chemical and Physical Investigations

The majority of glucose molecules in icodextrin are linearly linked withα (1-4) glucosidic bonds (>90%), while a small fraction (<10%) arelinked by a (1-6) bonds. Molecular weight distribution of icodextrin wasperformed by gel permeation chromatography. The distribution of α(1→6)and α(1→4) glucosidic links in icodextrin was assessed by nuclearmagnetic resonance spectroscopy. Volatile and semi-volatile organicimpurities were examined by high performance liquid chromatography andmass spectroscopy.

Dialysate Effluent Analysis

Dialysate effluent samples from patients were analyzed for icodextrinmetabolites, triglycerides, total protein and selective pyrogeniccytokines (IL-6, IL-1β and TNF-α). Icodextrin metabolites were assayedusing high performance anion exchange chromatography with pulsedamperometric detection. See, for example, Burke R A, Hvizd M G, ShockleyT R: Direct determination of polyglucose metabolites in plasma usinganion-exchange chromatography with pulsed amperometric detection, JChromatogr B 1997; 693:353-7. Triglyceride and protein analysis wereperformed with Boehringer Mannheim/Hitachi 911 Chemistry analyzer.Cytokine measurements were made using ELISA kits (R&D Systems,Minneapolis, Minn.).

Measurement of Pyrogens

Endotoxin concentration in icodextrin solution was determined by a fixedpoint chromogenic LAL test. See, for example, Weary M, Dubczak J,Wiggins J, et al: Validating an LAL chromogenic substrate pyrogen testfor large volume parenterals, In: Watson S W, Levin J, Novitsky T J,eds, Detection of bacterial endotoxin with limulus amebocyte lysatetest. New York: Alan R. Liss, 1987:307-22. Rabbit pyrogen test wasperformed in accordance with the guidelines provided in the EuropeanPharmacopeia. European Pharmacopoeia, Pyrogens, 4^(th) ed. Strasbourg,France: Council of Europe, 2002:131-2. An ex vivo pyrogen test thatmeasures IL-6 response in freshly isolated peripheral blood mononuclearcells (PBMC) following exposure to a test substance was used toquantitate non-endotoxin pyrogens. See, for example, Dinarello C A,O'Conner J V, LoPreste G: Human leukocyte pyrogen test for detection ofpyrogenic material in growth hormone produced by recombinant Escherichiacoli, J Clin Microbiol 1984; 20:323-9; and Poole S, Thorpe R, Meager A,et al: Detection of pyrogen by cytokine release, Lancet 1988; 1 (8577):130.

Measurement of Peptidoglycans

Peptidoglycan (PG) quantitation was performed with the silkworm larvaeplasma (SLP) test (Wako Pure Chemical Industries, Ltd., Osaka, Japan).See, for example, Tsuchiya M, Asahi N, Suzouki F: Detection ofpeptidoglycan and B-glucan with silkworm larvae plasma test, FEMSImmunol Medical Micrbiol 1996; 15:129-34; and U.S. Pat. No. 4,970,152.SLP contains all the factors of the prophenol oxidase (PPO) cascade, aself-defense mechanism of insects. The PPO cascade is initiated bypeptidoglycan, in which PPO is ultimately activated to phenol oxidase.The phenol oxidase activity is colorimetrically detected with3,4-dihydroxyphenylalanine as a substrate. The limit of detection ofpeptidoglycan in icodextrin solution was found to be 7.4 ng/mL. The SLPtest does not detect endotoxins.

As fully disclosed in U.S. Pat. No. 4,970,152, for example, thedetection of peptidoglycan (or □-G) can be carried out as follows. Asample containing the peptidoglycan is well mixed with a reagent thatincludes a fraction which specifically reacts with peptidoglycan (“PG”)to prepare a reaction solution. After a certain period of time, anenzymatic activity, e.g., activity of BAEEase, PPAE, PO, etc., in thereaction solution can be measured by a conventional method and comparedwith calibration curves previously obtained by using PG standardsolutions with known concentrations to determine the amount of PG.

Alternatively, it is possible to apply a phenomenon that a time requiredfor the activation of PO depends on the concentration of PG in thesample. That is, after mixing the PG reagent with a sample in thepresence of the substrate of PO, a time required for reaching a certainvalue of the amount of reaction product generated by PO is measured.

By way of example and not limitation, an experimental procedure thatutilizes the SLP test can be conducted in accordance with an embodimentof the present invention as follows.

Peptidoglycan (PG) and (1,3)-D-glucan (BG) are components of the cellwalls of gram positive bacteria and fungi, respectively. PG and BG aremeasured with the Silkworm Larvae Plasma (SLP) test. The SLP testcontains all the factors of the pro-phenol oxidase (PPO) cascade, animportant self-defense mechanism of insects. The PPO cascade isinitiated by peptidoglycan, in which PPO converts3,4-dihydroxyphenylalanine (DOPA) to melanin. The resulting formation ofmelanin is colorimetrically detected at 650 nm using a standard platereader. The raw material icodextrin is tested undiluted and has a limitof detection (LOD) of 0.74 ng/ml (the lowest detectable standard point).The finished product, such as EXTRANEAL, is tested after dilutingtenfold in order to mitigate the matrix inhibition caused by thepresence of electrolytes. The sample dilution step results in a LOD of7.4 ng/ml after correcting for the 1:10 dilution.

Animal Studies

No Observed Effect Level (NOEL) for peptidoglycan in icodextrin wasdetermined in a rat model. A total of 45 male Sprague-Dawley rats(Harlan Inc; Indianapolis, Ind., USA), weighing 255-280 g were dividedinto 9 equal groups. Each group received a single intraperitonealinjection of icodextrin spiked with 0, 1, 5, 10, 50, 100, 500, 1000 or5000 ng/mL peptidoglycan at a dose of 35 mL/kg. Peptidoglycan derivedfrom Staphylococcus aureus (Toxin Technology Inc., Sarasota, Fla., USA)was used in these experiments. Six hours post injection, the rats weresacrificed, and the fluid in the peritoneal cavity was quantitativelycollected by determining the weight and volume. Peritoneal fluid wasanalyzed for nucleated cell counts and differential counts, totalprotein, and IL-6 and TNF-α concentrations.

Statistical Analysis

Post market surveillance data on several hundred batches of icodextrinmanufactured between July 2000 and September 2002 were analyzed todetermine an association between peptidoglycan level and the incidenceof aseptic peritonitis. To accumulate sufficient complaint frequenciesto effectively display complaints per million (CPM), data wereclassified into peptidoglycan level in a log scale, namely,≦7.4, >7.4-15, >15-30, >30-60, and ≧60 ng/mL. For each of peptidoglycanconcentration range, the total complaints and the total units sold werecalculated. The CPM units sold were computed as the total complaintsdivided by the total units sold multiplied by one million. Negativebinomial regression was used to assess the association between CPM andpeptidoglycan level. The statistical analysis was performed using theSAS procedure GENMOD (SAS Institute, Cary, N.C., USA).

Clinical Cases

According to reports received through the BAXTER HEALTHCARECORPORATION's global pharmacovigilance system in 2000, the frequency[(number of complaints÷number of patients treated)×100] of asepticperitonitis was 0.095%. There was a steady increase in reported cases in2001 to a peak frequency of 1.04% in March 2002. The patients wererarely febrile or toxic in appearance; abdominal pain was modest toabsent; the dialysate was cloudy and cellular with dialysate cell countsvarying from 300 to 3500/mm³ (variable percentages of neutrophils,lymphocytes, and macrophages without eosinophils). Unlike bacterialperitonitis, there was no change in ultrafiltration or peritonealpermeability for small solutes. Blood and dialysate cultures wereinvariably negative. Antibiotics were variably instituted by the localphysicians. All clinical signs resolved upon stopping icodextrin. Insome cases, icodextrin was restarted, and aseptic peritonitis recurred.BAXTER HEALTHCARE CORPORATION initiated a voluntary worldwide recall ofseveral hundred batches of icodextrin in May 2002.

Chemical and Physical Investigations of Icodextrin

Extensive chemical and physical investigations of recalled icodextrinbatches were performed. These analyses did not reveal any differences inmolecular weight distribution of icodextrin, percent branching ofglucosidic bonds, or trace volatile and semi-volatile organic impuritiesbetween the batches associated with aseptic peritonitis and those notassociated with adverse clinical events. All solution components ofcomplaint batches of icodextrin-containing dialysate were within theproduct specifications and met current Pharmacopoeia standards.

Dialysate Effluent Analysis

A marked elevation in IL-6 concentration was observed in the dialysateeffluent from a patient with aseptic peritonitis compared to controleffluent (>5000 versus 59 pg/mL, respectively). An increase in proteinconcentration was noted with the complaint sample compared to thecontrol (236 mg/dL versus 125 mg/dL, respectively). There was nodifference in icodextrin and its metabolites (glucose polymers withdegree of polymerization from 2 to 7) between the complaint and controlsamples. These results indicated that neither icodextrin nor itsmetabolites were the likely cause of aseptic peritonitis.

Pyrogen Analysis of Icodextrin

Endotoxin levels in all the icodextrin samples associated with asepticperitonitis were found to be within the product limit (<0.25 EU/mL) asdetermined by the LAL test. In rabbit pyrogen tests, there was noincrease in temperature with either complaint or non-complainticodextrin batches. However, an increase in IL-6 response in the invitro PBMC assay was observed with both complaint icodextrin-containingdialysate batches and the icodextrin raw material used to manufacturethe complaint batches of peritoneal dialysate as illustrated in Table 1below:

TABLE 1 IL-6 response of complaint and non-complaint batches oficodextrin in the PBMC assay. In this assay, an IL-6 response of greaterthan 500 pg/mL is considered a positive pyrogenic response. Donor 023Donor 022 Donor 011 Test Sample pg/mL pg/mL pg/mL Control Medium^(a) 4524 50 Positive Control^(b) 12,000 10,000 10,000 Negative Control^(c) 13092 150 Icodextrin (non-complaint) 330 350 110 Icodextrin (complaint)5,100 7,000 970 Icodextrin (complaint) 4,200 4,200 700 Icodextrin rawmaterial (complaint) 9,300 780 1,600 Icodextrin raw material (non- 91100 130 complaint) ^(a)Eagle minimum essential medium with supplementalcomponents, ^(b)Baxter experimental product, ^(c)Glucose containingstandard peritoneal dialysis solution

The IL-6 provoking substance in the complaint samples was unaffected inthe presence of polymyxin B, thus suggesting that it was not LPS. See,for example, Pool E J, Johaar G, James S, et al: Differentiation betweenendotoxin and non-endotoxin pyrogens in human albumin solutions using anex vivo whole blood culture assay, J Immunoassay 1999; 20:79-89. In afiltration experiment using a 30 kD molecular weight cut-off filter, thecontaminant producing the inflammatory response in the PBMC assay wasfound in the retentate, suggesting the molecular weight of the substancewas >30 kD, i.e. larger than icodextrin. The negative assays for LPS,but positive PBMC IL-6 response indicated that the likely cause ofaseptic peritonitis was a non-endotoxin, pyrogen contaminant inicodextrin raw material used to manufacture the final peritonealdialysis solution.

Peptidoglycan and Microbiological Analysis

Analysis of several hundred recalled batches of icodextrin indicatedthat 41% of the batches were contaminated with peptidoglycan that wasdetected by the SLP assay. Peptidoglycan concentrations ranged from thedetection limit of 7.4 ng/mL to 303 ng/mL. Because the manufacture oficodextrin from maltodextrin requires heat and acidification, amicrobiological investigation for the presence of fastidious organismswas undertaken. Early steps of icodextrin were found to be contaminatedwith a thermophilic, acidiophilic, gram positive organism,Alicyclobacillus acidocaldarius. Alicyclobacillus was the source of thecontaminating peptidoglycan. Heat and sterile filtration proceduresapplied to the near final product eliminated the bacteria, but not thepeptidogycan contaminants. A positive correlation was found betweenpeptidoglycan levels in icodextrin and the IL-6 response observed in thePBMC assay (See, FIG. 1). Substantial variability in the IL-6 responsewas seen between donors suggesting a range of sensitivities topeptidoglycan.

Animal Studies

The effects of intraperitoneal administration of peptidoglycan in ratswas investigated in order to establish a NOEL. This can be used toestablish a regulatory specification for icodextrin manufacturing. Totalprotein, white blood cells and neutrophils were elevated in theperitoneal fluid of icodextrin+peptidoglycan-treated rats compared tocontrol rats that received icodextrin without peptidoglycan.Infiltration of neutrophils in peritoneal fluid showed a dose dependentincrease with a NOEL at 100 ng/mL (See, FIG. 2). Both inflammatorycytokines showed dose dependent increases with NOEL values at 10 ng/mLfor TNF-α (See, FIG. 3) and 100 ng/mL for IL-6 (See, FIG. 4). The lowestNOEL for peptidoglycan was for TNF-α response which was 10 ng/mL.

Correlation Between Peptidoglycan and Aseptic Peritonitis

A positive correlation was found between peptidoglycan concentration inicodextrin and CPM as illustrated in Table 2 below:

TABLE 2 Correlation between Complaints Per Million (CPM) units sold andpeptidoglycan concentration in icodextrin solution Total Peptidoglycan(ng/mL) Total units sold complaints CPM per units sold^(a) ≦7.4 290664353 18.2 >7.4-15  835539 10 12.0 >15-30 349503 10 28.6 >30-60 231330 834.6 ≧60  415099 105 253.0 ^(a)Complaints Per Million (CPM) units soldwas computed as the total complaints divided by the total units soldmultiplied by one million.

The baseline CPM was 18.2, when peptidoglycan levels were below thedetection limit of 7.4 ng/mL. In contrast, the CPM was 252.9, whenpeptidoglycan levels were equal to or greater than 60 ng/mL. Theassociation between CPM and peptidoglycan was highly significant(p<0.0001).

Incidence of Aseptic Peritonitis Following Corrective Action

In May 2002, BAXTER HEALTHCARE CORPORATION recalled all batches oficodextrin contaminated with peptidoglycan at a concentration >10 ng/mL,as well as all lots that were not assayed for peptidoglycan at the timeof the recall. In addition, an internal regulatory specification forpeptidoglycan below the detection limit of the assay (<7.4 ng/mL) wasimplemented for product release. Routine, serial monitoring forpeptidoglycan and thermophilic bacteria was implemented. Thepeptidoglycan concentrations in all the recent batches manufactured havebeen below the detection limit of the SLP test and are devoid of an IL-6eliciting agent by the PBMC assay.

FIG. 5 shows the incidence rate of aseptic peritonitis by monthbeginning September 2001 to January 2003. The frequency of complaintshas decreased from a peak value of 1.04% in March 2002 to 0.013% inJanuary 2003 following the implementation of corrective actions. Thenumber of patients using a commercially-available peritoneal dialysissolution known as EXTRANEAL from BAXTER HEALTHCARE CORPORATION duringthis period remained at approximately 7,000. These results suggest thatcorrective actions have been effective in preventing excess complaintsof aseptic peritonitis due to peptidoglycan contamination inicodextrin-containing dialysis solution.

As previously discussed, the present invention relates to methods andcompositions that employ the detection of peptidoglycan in peritonealdialysis solutions. This allows testing for bacterial fragments aspeptidoglycans are the major cell wall component of gram positiveorganisms. In this regard, peptidoglycan detection can be effectivelyutilized to prevent peritonitis in patients that use the peritonealdialysis solutions, such as peritoneal dialysis solutions that contain aglucose polymer including icodextrin and the like.

It should be appreciated that compositions, peritoneal dialysissolutions and methods of manufacturing and using same can be provided inany suitable manner in accordance with an embodiment of the presentinvention. In an embodiment, the present invention provides bioburdentesting, such as bioburden testing for acidophilic thermophilicorganisms according to standard procedures as described in variousPharmacopoeias. This type of testing can be utilized as an added levelof testing in addition to the testing for bacterial fragments bydetection of peptidoglycans as previously discussed.

In an embodiment, the present invention provides methods formanufacturing a peritoneal dialysis solution. The method can include anysuitable number and type of processing stages. For example, the processincludes providing a glucose polymer; adding a reagent to the glucosepolymer wherein the reagent is capable of reacting with a peptidoglycan;determining an amount of the peptidoglycan; and using the glucosepolymer to make the peritoneal dialysis solution if it is determinedthat a sufficiently low level of the peptidoglycan is present in theglucose polymer. If the amount of peptidoglycan or the like exceeds thislevel, such as about 10 ng/mL or less, the glucose polymer can befurther processed to remove the peptidoglycan or the like in order toachieve a sufficiently low level of same. The glucose polymer can befurther processed in any suitable manner. In an embodiment, the glucosepolymer can be processed with any suitable number and type of separationdevices, such as an affinity device and/or the like.

In an embodiment, the present invention provides compositions, such asglucose polymer compositions that can be used to prepare peritonealdialysis solutions. A variety of different and type of compositions andsolutions containing same can be utilized. For example, the type ofcompositions and solutions can same can be found in U.S. Pat. No.4,761,237, entitled “PERITONEAL DIALYSIS SOLUTION CONTAININGCARBOHYDRATE POLYMERS; U.S. Pat. No. 4,886,789, entitled “PERITONEALDIALYSIS AND COMPOSITIONS FOR USE THEREIN”; U.S. Pat. No. 6,077,836,entitled “PERITONEAL DIALYSIS AND COMPOSITIONS FOR USE THEREIN”; ANDU.S. Pat. No. 6,248,726 B1, entitled “METHOD OF PERITONEAL DIALYSISUSING GLUCOSE POLYMER SOLUTIONS, the disclosures of which in theirentirety are incorporated herein by reference. Additional examples ofcompositions and solutions containing same can be can be found in U.S.patent application Ser. No. 10/327,264, entitled BIOCOMPATIBLE DIALYSISFLUIDS CONTAINING ICODEXTRINS, filed on Dec. 20, 2002; and U.S. patentapplication Ser. No. 09/206,063, entitled PERITONEAL DIALYSIS SOLUTIONCONTAINING MODIFIED ICODEXTRINS, filed on Dec. 4, 1998, the disclosuresof which in their entirety are incorporated herein by reference. In anembodiment, the peritoneal dialysis solutions can include EXTRANEAL byBAXTER HEALTHCARE CORPORATION or suitable modifications thereof.

In an embodiment, the present invention includes methods of providingperitoneal dialysis, such as continuous ambulatory peritoneal dialysisand automated peritoneal dialysis. In continuous ambulatory peritonealdialysis, the patient performs several drain, fill, and dwell cyclesduring the day, for example, about four times per day. Each treatmentcycle, which includes a drain, fill and dwell, takes about four hours.

Automated peritoneal dialysis is similar to continuous ambulatoryperitoneal dialysis in that the dialysis treatment includes a drain,fill, and dwell cycle. However, a dialysis machine automaticallyperforms three or more cycles of peritoneal dialysis treatment,typically overnight while the patient sleeps.

With automated peritoneal dialysis, an automated dialysis machinefluidly connects to an implanted catheter or the like. The automateddialysis machine also fluidly connects to a source or bag of freshdialysis solution and to a fluid drain. The dialysis machine pumps spentdialysis solution from the peritoneal cavity, through the catheter, tothe drain. The dialysis machine then pumps fresh dialysis solution fromthe source, through the catheter, and into the patient's peritonealcavity. The automated machine allows the dialysis solution to dwellwithin the cavity so that the transfer of waste, toxins and excess waterfrom the patient's bloodstream to the dialysis solution can take place.A computer controls the automated dialysis machine so that the dialysistreatment occurs automatically when the patient is connected to thedialysis machine, for example, when the patient sleeps. In this regard,the dialysis system automatically and sequentially pumps fluid into theperitoneal cavity, allows for dwell, pumps fluid out of the peritonealcavity, and repeats the procedure.

Several drain, fill, and dwell cycles will occur during the treatment.Also, a final volume “last fill” is typically used at the end of theautomated dialysis treatment, which remains in the peritoneal cavity ofthe patient when the patient disconnects from the dialysis machine forthe day. Automated peritoneal dialysis frees the patient from having tomanually perform the drain, dwell, and fill steps during the day. In anembodiment, automated peritoneal dialysis can be performed by utilizingan admix device, such as ADMIX HOMECHOICE by BAXTER HEALTHCARECORPORATION or suitable modifications thereof.

In an embodiment, the present invention provides a test to determine ifa peritoneal dialysis solution, such as an icodextrin-based solution,includes peptidoglycan that exceeds a level sufficient to causeperitonitis in the patient that uses same. The present inventionprovides a detection protocol that utilizes a reagent, such a reagentderived from a silkworm larvae plasma, for such purpose. It should beappreciated that the detection procedure can be carried out at anysuitable stage prior to use of the peritoneal dialysis solution.

For example, the reagent can be added to a glucose polymer composition,such as an icodextrin composition, in raw material form to determine thepresence of peptidoglycan. If the level of peptidoglycan is at asufficiently low level, the composition can then be utilized to preparethe peritoneal dialysis solution. In another embodiment, the reagent canbe added to the peritoneal dialysis solution, such as after thesterilization process in finished product form. For example, the reagentcan be added to the peritoneal dialysis solution that is containedwithin any suitable solution bag, such as a single-chambered solutionbag or a multi-chamber solution bag. An example of a multi-chambersolution bag or container is provided in U.S. Pat. No. 5,431,496, thedisclosure of which in its entirety is incorporated herein by reference.

As described herein, all patent applications and publications includingpatent publications are incorporated herein by reference in theirentirety.

It should be understood that various changes and modifications to thepresently preferred embodiments described herein will be apparent tothose skilled in the art. Such changes and modifications can be madewithout departing from the spirit and scope of the present invention andwithout diminishing its intended advantages. It is therefore intendedthat such changes and modifications be covered by the appended claims.

The invention is claimed as follows:
 1. A method for manufacturing adialysis solution, the method comprising: providing a glucose polymer;performing a modified bioburden test on the glucose polymer to detectcontaminating Alcyclobacillus acidocaldarius; testing the glucosepolymer with a reagent that is reactive with any peptidoglycan in theglucose polymer to determine a peptidoglycan concentration; and usingthe glucose polymer to prepare the dialysis solution if thepeptidoglycan concentration is determined to be about 10 ng/mL or less.2. The method of claim 1 further comprising testing the dialysissolution with a reagent that is reactive with any peptidoglycan in thedialysis solution to determine if the peptidoglycan concentration in thedialysis solution is less than about 10 ng/mL.
 3. The method of claim 1,wherein the reaction with the reagent initiates a serine proteasecascade.
 4. The method of claim 1, wherein the peptidoglycanconcentration is determined by a colorimetric measurement.
 5. The methodof claim 1, wherein the modified bioburden test includes testing for theAlicyclobacillus acidocaldarius at a pH of less than about 5.0.
 6. Themethod of claim 1, wherein the modified bioburden test includes testingfor the Alicyclobacillus acidocaldarius at a temperature of 50° C. to60° C.
 7. The method of claim 1, wherein the glucose polymer includesicodextrin.
 8. A method of testing a glucose polymer for peptidoglycancontamination, the method comprising: providing a glucose polymer;performing a modified bioburden test on the glucose polymer to detectAlicyclobacillus Acidocaldarius, which is a source of peptidoglycan; andtesting the glucose polymer with a reagent that is reactive with anypeptidoglycan in the glucose polymer to determine a peptidoglycanconcentration in the glucose polymer.
 9. The method of claim 8 furthercomprising preparing a dialysis solution including the glucose polymerand testing the dialysis solution with a reagent that is reactive withany peptidoglycan in the dialysis solution to determine if thepeptidoglycan concentration in the dialysis solution is less than about10 ng/mL.
 10. The method of claim 8, wherein the reaction with thereagent initiates a serine protease cascade.
 11. The method of claim 8,wherein the serine protease cascade includes a prophenol oxidasecascade.
 12. The method of claim 8, wherein the modified bioburden testincludes testing for the Alicyclobacillus acidocaldarius at a pH of lessthan about 5.0.
 13. The method of claim 8, wherein the modifiedbioburden test includes testing for the Alicyclobacillus acidocaldariusat a temperature of 50° C. to 60° C.
 14. The method of claim 8, whereinthe glucose polymer includes icodextrin.
 15. A method of providingdialysis to a patient, the method comprising: preparing a dialysissolution including: providing a glucose polymer; performing a modifiedbioburden test on the glucose polymer to detect contaminatingAlcyclobacillus acidocaldarius; testing the glucose polymer using areagent that is reactive with peptidoglycan to determine a peptidoglycanconcentration; and using the glucose polymer to prepare the dialysissolution if the peptidoglycan concentration is determined to be about 10ng/mL or less; and administering the dialysis solution to the patient.16. The method of claim 15 further comprising testing the dialysissolution with a reagent before administering the dialysis solution tothe patient, wherein the reagent is reactive with any peptidoglycan inthe dialysis solution to determine if the peptidoglycan concentration inthe dialysis solution is less than about 10 ng/mL.
 17. The method ofclaim 15, wherein the dialysis solution is a peritoneal dialysissolution.
 18. The method of claim 15, wherein the dialysis solution isprovided during peritoneal dialysis selected from the group consistingof an automated peritoneal dialysis and a continuous peritonealdialysis.
 19. The method of claim 15, wherein the modified bioburdentest includes testing for the Alicyclobacillus acidocaldarius at a pH ofless than about 5.0.
 20. The method of claim 15, wherein the modifiedbioburden test includes testing for the Alicyclobacillus acidocaldariusat a temperature of 50° C. to 60° C.